In abundant food, C. elegans develops through four distinct larval stages (L1-L4) to the adulthood. However, when conditions become less favorable, the development is arrested and an alternative third-stage larvae is formed which is specialized for dispersal and long-term survival, termed dauer. Dauer larvae don't feed, are long-lived and resistant to stress. Morphologically, they can be distinguished from adults because they are thinner, darker, and have a constricted pharynx. The changes in morphology correlate with dramatic alterations in the expression pattern of genes in dauers and adults. (Riddle, 1988; Riddle and Albert, 1997)
In the past, temperature-sensitive strains have been identified that are dauer-constitutive; e.g., at the restrictive temperature of 25° C. these strains form dauers even in the presence of food (Gems, 1998). It turns out that many of these strains, termed daf strains, have acquired mutations in genes involved in the nematode insulin/IGF-1 signaling pathway. Studies of the phenotypes have allowed certain daf genes to be ordered into a genetic pathway consisting of DAF-2/IR, age-1/PI-3 Kinase, pdk-1, akt-1, akt-2, and the FOXO transcription factor DAF-16 (Gottlieb and Ruvkun, 1994; Riddle, 1977; Riddle et al, 1981, Kaestner et al., 2000).
It has been shown by Northern blotting and RT-PCR that the expression of the sod-3 gene is regulated by mutations in the DAF-2/insulin receptor pathway (Honda & Honda, 1999 ). Inactivation of the DAF-2 function in certain mutant strains results in a strong up-regulation of the sod-3 expression. Honda & Honda suggested that DAF-16 is the transcription factor activating the sod-3 gene and that DAF-16 is inhibited by the DAF-2/IR pathway.
Furthermore, a consensus sequence binding to the transcription factor DAF-16 has been identified and this sequence was shown to be present in the sod-3 upstream regulatory region (Furuyama et al., 2000). This binding motif fused to a minimal promoter was sufficient for insulin-regulated expression in mammalian tissue culture systems.
Since the DAF-2/insulin receptor pathway and its components are very well conserved in man, it was proposed to use the dauer phenotype to identify modulators of the insulin/IGF-1 signaling in man (WO 98/51351 A1). However, the assay systems according to the prior art require long incubation times until the developmental program of the dauer larvae has been completed (usually 3-5 days). Such a long time period may result in the degradation of the assay components. Moreover, the impermeable cuticula structure of dauers together with the reduced food-intake might be a setback for compound uptake into the worm.
Therefore, it was the underlying problem of instant invention to provide a process for the identification of compounds that modulate the DAF-2/IR pathway, which does not depend on C. elegans dauer larvae and overcomes the above-mentioned disadvantages. The process of the invention (i.e., the assay system of the invention) relies on a data read-out that is directly linked to the DAF-2/IR pathway, and which is not influenced by the progress of developmental stages of the organism under investigation, preferably mammalian and nematode cells, particularly nematode cells, e.g., C. elegans. Furthermore, the assay should provide quantitative data read-out after a short incubation time, preferably within about 8-12 hours, in the presence of the compound(s) to be investigated. Depending on the reporter used in the assay, a quantitative data read-out is obtainable in contrast to the prior art assay systems.